*Protein ID (NP or XP #) or Wolbachia#: NP_220958.1
*Organism (including strain):Rickettsia prowazekii, str. Madrid E
Etiologic Risk Group (see link below): Category B priority pathogen, National Institute of Health
*/Disease Information (sort of like the Intro to your Mini Research Write up): Rickettsia prowazekii is part of the family of Rickettsia bacteria, all of which are responsible for Typhus and different strains of spotted fever. R. prowazekii in particular is responsible for epidemic typhus fever, an infectious disease characterized by symptoms such as high fever, head aches, muscle aches, and fatigue. In severe cases, symptoms can even include life-threatening symptoms such as encephalitis or kidney failure. While epidemic typhus is most prevalent in the cold, mountainous, regions of Asia, Central Africa, South and Central America, infections have also been seen in the United States. In addition, R. prowazekii is a seen as a possible bioterrorism threat due to the risk for it to be contracted via inhalation in an aerosolized form. The bacterium is transmitted through the feces of human body lice, which have been infected with R. prowazekii after feeding on another individual infected with the disease. When the feces are pushed into the skin, through an open lesion such as the bite site, the bacteria are able to enter the body and cause infection.
Link to TDR Targets page (if present): no TDR targets page
-- PDB # or closest PDB entry if using homology model: 3D53
-- For Homology Model option: homology model in E. coli (with substrate in active site) 2AU7
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB ---- Query Coverage: 82%
---- Max % Identities: 68%
---- Chain used for homology: A
Current Inhibitors: No inhibitors have been identified yet
Expression Information (has it been expressed in bacterial cells): RpIPP has not yet been expressed in bacterial cells
Purification Method: Ni-NTA chromatography
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MFIKKIKAKANNNEINVIIEIPMNSGPIKYEFDKESGALFVDRFMQTTMSYPCNYGFIPDTLSNDGDPVDV LVVAHHPVVPGSVIKCRAIGVLMMEDESGLDEKIIAVPTSKLDITFDHIKELDDLCEMLKKRIVHFFEHYKD LEKGKWVKVTGWGDKVKAETLIKEGIDRN
*length of your protein in Amino Acids: 173
Molecular Weight of your protein in kiloDaltons using the **Expasy ProtParam** website: 42221.2 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 4250 M-1cm-1
*NCBI Gene # or RefSeq#: 883528
*Protein ID (NP or XP #) or Wolbachia#: NP_220958.1
*Organism (including strain): Rickettsia prowazekii, str. Madrid E
Etiologic Risk Group (see link below): Category B priority pathogen, National Institute of Health
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Rickettsia prowazekii is part of the family of Rickettsia bacteria, all of which are responsible for Typhus and different strains of spotted fever. R. prowazekii in particular is responsible for epidemic typhus fever, an infectious disease characterized by symptoms such as high fever, head aches, muscle aches, and fatigue. In severe cases, symptoms can even include life-threatening symptoms such as encephalitis or kidney failure. While epidemic typhus is most prevalent in the cold, mountainous, regions of Asia, Central Africa, South and Central America, infections have also been seen in the United States. In addition, R. prowazekii is a seen as a possible bioterrorism threat due to the risk for it to be contracted via inhalation in an aerosolized form. The bacterium is transmitted through the feces of human body lice, which have been infected with R. prowazekii after feeding on another individual infected with the disease. When the feces are pushed into the skin, through an open lesion such as the bite site, the bacteria are able to enter the body and cause infection.
Link to TDR Targets page (if present): no TDR targets page
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): https://www.patricbrc.org/portal/portal/patric/Feature?cType=feature&cId=PATRIC.272947.5.NC_000963.CDS.740735.741253.fwd
Essentiality of this protein: Found to be essential in E.coli
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): multimer
Complex of proteins?: Yes, six chains
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): https://www.doria.fi/bitstream/handle/10024/69593/AnnalesAI417Tuominen.pdf?sequence=1
*EC#: 3.6.1.1.
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=3.6.1.1
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Colorimetric Assay
-- link to Sigma (or other company) page for assay (see Sigma links below) http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/ionorgpyrophosph.pdf
-- links to assay reagents (substrates) pages.
TRIS buffer: https://www.sigmaaldrich.com/Graphics/Supelco/objects/4800/4709.pdf
Sodium Pyrophosphate: http://www.sigmaaldrich.com/catalog/product/sial/221368?lang=en®ion=US
Magnesium Chloride solution: http://www.sigmaaldrich.com/catalog/product/sigma/m1028?lang=en®ion=US
Ammonium molybdate solution: http://www.sigmaaldrich.com/catalog/product/aldrich/277908?lang=en®ion=US
--- List cost and quantity of substrate reagents, supplier, and catalog #
TRIS buffer: 100g/ $36.00, Sigma Aldrich 252859
Sodium Pyrophosphate: 100g/ $31.60, Sigma Aldrich 221368
Magnesium Chloride solution: 100ml/ $50 or 100g powder/ $30.30, Sigma Aldrich M1028/ 208337
Ammonium Molybdate: 5g/ $75.80, Sigma Aldrich 277908
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 3D53
-- For Homology Model option: homology model in E. coli (with substrate in active site) 2AU7
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 82%
---- Max % Identities: 68%
---- Chain used for homology: A
Current Inhibitors: No inhibitors have been identified yet
Expression Information (has it been expressed in bacterial cells): RpIPP has not yet been expressed in bacterial cells
Purification Method: Ni-NTA chromatography
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MFIKKIKAKANNNEINVIIEIPMNSGPIKYEFDKESGALFVDRFMQTTMSYPCNYGFIPDTLSNDGDPVDV
LVVAHHPVVPGSVIKCRAIGVLMMEDESGLDEKIIAVPTSKLDITFDHIKELDDLCEMLKKRIVHFFEHYKD
LEKGKWVKVTGWGDKVKAETLIKEGIDRN
*length of your protein in Amino Acids: 173
Molecular Weight of your protein in kiloDaltons using the **Expasy ProtParam** website: 42221.2 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 4250 M-1cm-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): ATGTTCATCAAAAAGATCAAAGCGAAAGCTAACAACAACGAAATCAACGTTATCATCGAA
ATCCCGATGAACTCTGGTCCGATCAAATATGAATTCGATAAAGAATCTGGCGCGCTGTTC
GTTGACCGTTTCATGCAGACCACCATGTCTTACCCGTGCAACTACGGTTTCATCCCGGAC
ACCCTCTCTAACGACGGTGACCCGGTTGACGTTCTGGTTGTTGCGCACCACCCTGTTGTT
CCGGGTTCTGTTATCAAATGCCGTGCGATCGGTGTTCTGATGATGGAAGACGAGTCTGGT
CTCGACGAGAAAATCATCGCGGTTCCGACCTCTAAACTGGACATCACCTTCGACCACATC
AAAGAGCTGGACGACCTGTGCGAAATGCTGAAAAAACGTATCGTTCACTTCTTCGAACAC
TACAAAGACCTGGAAAAAGGTAAATGGGTTAAAGTTACCGGTTGGGGTGACAAAGTTAAA
GCGGAAACCCTGATTAAAGAGGGTATCGACCGTAACTAA
*GC% Content for gene (codon optimized): 42.1%